1. Field of the Invention
The present invention generally relates to three-dimensional scanning with a light beam such as a laser. More particularly, the invention relates to high speed microscopy with three-dimensional laser beam scanning.
2. Description of Related Art
In experimental biology, increasing numbers of studies that are carried out on the cellular or sub-cellular level apply optical imaging techniques for combined structural and functional analysis of live nerve cells. Optical imaging approaches reduce the need for both post-experiment histology and invasive micropipette techniques during the experiment by enabling direct visualization of fine structures of interest and measurement of their function by means of fluorescent indicators, e.g. molecular or genetically encoded probes. Specifically in experimental neuroscience, optical techniques to probe membrane potential and intracellular ion concentrations of visually identified nerve structures have become a powerful tool to investigate many aspects of brain function. However, the requirements for functional imaging of living nerve cells demand imaging systems with spatial resolutions in the low to sub-micrometer range and frame rates of up several 1,000 per second.
Advanced imaging schemes such as confocal microscopy and multiphoton microscopy are increasingly employed in experimental neuroscience, since they significantly reduce light scattering related degradation in image quality, which is of particular importance in mammalian brain tissue. This property supports optically sectioning living biological specimens for computer-based three-dimensional reconstruction. While both microscope principles utilize point illumination from a laser source focused into a small spot, they differ in their concepts of fluorescence excitation and image formation. Confocal microscopy employs visible laser light for single-photon excitation and obstructs out-of-focus and scattered fluorescence by spatial filtering of the resulting image with a pinhole that is confocal with the illumination spot. Multiphoton microscopy uses infrared (IR) laser light, which is inherently less scattered due to its longer wavelength. Because of its lower energy, multiple photons have to be simultaneously absorbed to excite a single fluorescent molecule. This non-linear dependence on intensity limits the fluorescence excitation to a very small focal volume (rendering spatial filtering unnecessary as needed for confocal microscopy). In order to achieve a useful probability of multiphoton absorption events, extremely high photon flux is needed, which is achieved by the use of lasers emitting ultra-fast pulses in the high femtosecond (fs) to low picosecond (ps) range with accordingly high peak power, while maintaining biologically tolerable average power in the low milliwatt range (mW) at the preparation level.
With both of these schemes, the laser beam is commonly raster-scanned to obtain an image. The scanning principle generally employed is mechano-optical, i.e. galvanometer-driven mirrors; thus, inertia limits the speed of such systems. Therefore, while confocal and multi-photon microscopy have proven to be extremely useful in obtaining structural images, success of these techniques with functional imaging such as optical recording of neuronal activity has been limited, as neither raster-scanning nor random-access of multiple sites-of-interest is possible at the required rates. While video-rate imaging with multiphoton microscopy has been documented using resonant galvanometers or micro-lens arrays, neither of these support high-speed functional imaging. In general, existing confocal and multi-photon microscopes lack the spatio-temporal resolution necessary to measure fast multi-site optical signals from small brain structures. With these microscopes, the only possibility to achieve high frame rates necessary for functional imaging is to scan fewer or even single lines. Due to these constraints in scan patterns most microscopes cannot follow complex shaped biological structures such as neuronal dendrites.
In addition to the limited flexibility of lateral scan pattern, fast functional imaging of three-dimensional structures is practically impossible due to the low focusing speed of microscopes. The fastest and most precise change of focal plane can be obtained by piezo-actuators that are inserted between the microscope body and the objective lens. However, even these linear motors require axial step times (>10 ms) that are significantly longer per change of focus plane than the total time available to scan the entire volume-of-interest (<1 ms) containing many such planes. Even if faster methods to axially position the objective lens were available, resulting complications such as shock waves in the immersion solution would make these attempts fail because of movement artifacts interfering with delicate living tissue and patch-clamp seals.
Multiphoton laser scanning microscopy (MPLSM) has revolutionized fluorescence imaging in the field of biology by enhancing the quality of images obtained from optically thick tissue. Some of the most productive applications of MPLSM have been in the field of experimental neuroscience, where it has been used to study neurodegenerative diseases, synaptic plasticity, and neuronal integration in optically scattering live brain tissue. While the inherent sectioning ability, in combination with low levels of photodamage, provided by MPLSM has made it extremely useful for the structural imaging of small processes (e.g., dendritic spines ˜1 um) deep within neuronal tissues, the low temporal resolution available in most commercial MPLSM systems has limited its applicability in functional imaging to studies that involve only a few sites of interest. This is mainly because the maximum speed of the commonly used galvanometer-based method of laser scanning is inherently limited by inertia. Therefore, in order to achieve the sampling speeds necessary for monitoring physiological signals such as intracellular calcium dynamics, users typically restrict their scan region to a single line. This not only significantly reduces the ability to accurately track physiological signals in complex shaped cells such as neurons, but also prevents the monitoring of signals from more than just a few sites of interest along a neuron.
In order to overcome this limitation, systems have been developed which combine the enhanced image quality provided by multiple advanced imaging techniques, including MPLSM, with the enhanced temporal resolution and scanning flexibility of acousto-optic deflectors (AODs) to enhance the study of neurophysiological processes. AODs utilize high frequency acoustic waves that are propagated in an acousto-optic (AO) medium as a tunable diffraction grating. Adjusting the frequency of the acoustic wave changes the diffraction angle and results in inertia-free beam deflection. By utilizing this property of AODs, these systems have been able to lower transition times between sites on a specimen to values in the low microsecond range (˜20 μs) and remove any limit to the dwell time at a site. AODs have also allowed users to sample from any site in the specimen plane with the same transition time. As a result, it is possible to effectively monitor calcium transients along extensive regions of a neuron located deep in optically thick brain slices, a task which can not be performed with available commercial galvanometer-based confocal or multi-photon systems.
However, despite these enhancements in temporal resolution, the ability to effectively monitor signals at physiological speeds is limited to two-dimensional (2D) scans. This is because the inertia-free beam steering properties of AODs have thus far only been applied to lateral scanning while the axial position of the focus is adjusted by the same mechanism available in most commercial systems, i.e. raising or lowering the objective lens with an actuator. This moves the back focal aperture of the objective lens and, since the focal length of the objective lens remains constant, also moves the axial position of the focus by the same amount. Thus, the axial speed of laser scanning is inherently limited by the inertia of the objective lens and its actuator. Consequently, the highest speed at which the axial position of the focus can be moved, even with the fastest available commercial equipment commonly available (piezo-actuators capable of 10 ms per step), is still orders of magnitudes slower than the speed at which we are able to move the lateral position of the focus. This inability to effectively monitor physiological signals in three dimensions is a serious constraint in neuroscience, since neurons are complex three dimensional (3D) structures and develop in 3D networks.
There are different methods that have been proposed which would allow for laser scanning in the axial dimension. One of the most developed is the variable focal-length liquid-filled lens. The principal mechanism for changing the focal length within this type of lens relies on changing the pressure within a lens chamber, which in turn deforms an elastic membrane and changes the curvature of the lens. Recent developments have increased the maximum numerical aperture and the speed at which the focal position can be changed, but despite these improvements, the fastest variable focal length liquid-filled lenses typically still require at least one millisecond to change the focal position. Variable focal length lenses which use nematic liquid crystals or electro-optic materials to change the refractive index of the lens rather than the shape have also been developed. In general, for these types of lenses, generating fast response times requires restricting the thickness of the lens cell, which in turn severely limits the maximum change in focal length. Other mechanisms for variably adjusting focal length include using a deformable mirror, however this method also requires at least a millisecond to change the focal position and therefore is not suitable for applications that require frame rates greater than 1 kHz.